WO2003080209A2 - Method of concentrating macromolecules or agglomerates of molecules or particles - Google Patents

Method of concentrating macromolecules or agglomerates of molecules or particles Download PDF

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Publication number
WO2003080209A2
WO2003080209A2 PCT/FR2003/000920 FR0300920W WO03080209A2 WO 2003080209 A2 WO2003080209 A2 WO 2003080209A2 FR 0300920 W FR0300920 W FR 0300920W WO 03080209 A2 WO03080209 A2 WO 03080209A2
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WIPO (PCT)
Prior art keywords
macromolecule
agglomerate
dna
concentration
liquid sample
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PCT/FR2003/000920
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French (fr)
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WO2003080209A3 (en
Inventor
Jean Berthier
Laurent Davoust
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Commissariat A L'energie Atomique
Centre National De La Recherche Scientifique
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Application filed by Commissariat A L'energie Atomique, Centre National De La Recherche Scientifique filed Critical Commissariat A L'energie Atomique
Priority to JP2003578029A priority Critical patent/JP4391829B2/en
Priority to US10/507,521 priority patent/US20050181367A1/en
Priority to DE60308338T priority patent/DE60308338T2/en
Priority to EP03727612A priority patent/EP1488004B1/en
Publication of WO2003080209A2 publication Critical patent/WO2003080209A2/en
Publication of WO2003080209A3 publication Critical patent/WO2003080209A3/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption

Definitions

  • the present invention relates to a process for the concentration of macromolecules or agglomerates of molecules or particles, with a view to possible detection or specific extraction of said macromolecules or said agglomerates.
  • the techniques for concentrating macromolecules or agglomerates of molecules are, for the most part, intended for the field of medical diagnosis, in particular for the detection of DNA strands or protein complexes such as antigens or prions.
  • the current technique consists, most often, of concentrating DNA strands by amplification in a liquid medium, according to the technique commonly designated by the abbreviation PCR (“Polymerase Chain Reaction” or “ Polymerase Chain Reaction ”).
  • This technique consists in replicating the DNA strands contained in a liquid sample a large number of times (up to 10 5 -10 6 times) by injecting polymerase into the sample subjected to a series of thermal cycles.
  • the concentration in DNA is large enough to allow detection.
  • this technique requires a significant duration, because of the consequent number of thermal cycles to be reproduced in order to have a sufficient quantity of DNA.
  • this technique is accompanied by a significant background noise because the polymerase can amplify DNA segments present in the liquid sample, which are different in nature from the DNA segments to be detected.
  • the antigen being sandwiched between two labeled antibodies for example.
  • These tests have the advantage of being rapid and inexpensive. They are the ones that are used extensively in broadband screening programs in the pharmaceutical industry, for example. However, their sensitivity is limited, around 1 nM with regard to tests based on nucleic acids. This is why the diagnostic industry preferably uses tests in heterogeneous phase, in which an antibody is immobilized on a solid support, support which it will then be possible to wash in order to remove almost all of the labeling reagents. In addition, an amplification of the signal is carried out, using an enzyme as a labeling molecule.
  • Prions responsible in particular for spongiform encephalopathies, are complexes or agglomerates made up of a natural protein, the glycoprotein PrP c , normally present on the surface of many cells in the body and of an infectious protein PrP sc , which does not differs from normal PrP c glycoprotein only by its conformation, which is abnormally folded.
  • PrP sc proteins are able, on the one hand, to associate with PrP c proteins and on the other hand, able to induce the transformation of normal proteins into infectious proteins. Detection of prions is made difficult by the fact that they are present in notable quantities only in the brain, whereas they are found in traces in the blood.
  • the PMCA technique thus makes it possible to concentrate the prions in the sampling medium, that is to say the blood, so that they can be detected. To do this, a sonication process intended to fragment the prions is implemented. All the prions resulting from this ultrasonic fragmentation regrow in-vitro using the PrP c proteins in the sample. This elementary cycle (fragmentation-regrowth) is repeated as many times as necessary, until the quantity of prions becomes detectable.
  • the detection step facilitated by the amplification step, is carried out by fluorescence spectroscopy.
  • the proteins to be detected are labeled using a fluorescent probe, the presence of which is manifested when this is illuminated by light of characteristic wavelength.
  • this technique generally generates a significant background noise, since the probes can associate with other molecules than the prions to be detected.
  • a more elaborate technique known as the fluorescence correlation technique, uses two fluorescent probes which emit at two different wavelengths and which both hang symmetrically and specifically on PrP c proteins, the latter being in significant concentration within prions.
  • a very small volume of the liquid containing the prions to be detected is then illuminated with beams at two different wavelengths, and the two intense fluorescent emissions due to the presence of prions, which constitute an agglomerate of normal or infectious PrP proteins on which the fluorescent probes are attached.
  • background noise remains, due in particular to the presence of fluorescent probes which could not find specific adsorption sites and to the presence of normal PrP c proteins isolated, to which fluorescent probes have become attached.
  • the object of the present invention is precisely to propose a process for the concentration of macrolecules or agglomerates of molecules or particles, which allows in particular a selective concentration of said macromolecules or said agglomerates, with a view to possible detection by limiting as much as possible the background noise, or for possible purification of a sample containing said macromolecules or said agglomerates.
  • the subject of the present invention is a process for the selective concentration of a macromolecule or an agglomerate of molecules or particles contained in a liquid sample, successively comprising the following steps:
  • interfacial layer a monolayer (or almost two-dimensional area) located on the surface of the liquid sample (called first liquid phase) comprising the macromolecule or the agglomerate to be concentrated. Due to its nature and specific properties, this layer is capable of ensuring the selective transfer of the macromolecule or of the agglomerate from the liquid sample to the interfacial layer, and, because of its minute volume compared to the sample liquid, to concentrate said macromolecule or said agglomerate.
  • the interfacial layer may correspond to a second liquid phase deposited on the surface of the liquid sample (case for example, when it is a question of concentrating a macromolecule of the DNA type), this phase having characteristics such that it makes it possible to attract the macromolecule or the agglomerate towards it.
  • the interfacial layer can also correspond to the interface between the ambient atmosphere and the liquid sample (for example, when the macromolecule or the agglomerate to be concentrated has a hydrophobic character, such as prions, and when the liquid sample containing is an aqueous medium).
  • the method of the invention may comprise a prior step (located before the step of forming the dispersion) consisting in depositing said second on the surface of the liquid sample liquid phase or interfacial layer.
  • the step of forming the dispersion is carried out by mechanical agitation of the medium comprising the liquid sample and the interfacial layer or by injection directly into the liquid sample surmounted by the interfacial layer of gaseous or liquid capillary jets.
  • a foam designates a dispersion comprising a set of gas bubbles (typically air) coexisting with an interstitial liquid medium, in the form of thin interstitial films between the bubbles.
  • This type of dispersion thus provides a multitude of liquid-gas interfaces.
  • the foam according to the invention can be obtained, for example, by vigorous mechanical agitation of the abovementioned medium or by injection into this medium of gaseous capillary jets (typically air jets).
  • an emulsion designates a dispersion, in which the interfacial layer is divided into globules within the liquid sample, said liquid sample constituting an interstitial medium. A multitude of liquid-liquid interfaces is thus formed, and the contact surface between the liquid sample and the interfacial layer is considerably increased.
  • the emulsion in the same way as the foam, can be obtained by mechanical agitation of the medium comprising the liquid sample and the interfacial layer but also by direct injection into the liquid sample surmounted by the interfacial layer of liquid or gaseous capillary jets.
  • the fact of going through a dispersion of the foam or emulsion type to ensure the concentration of a macromolecule or of an agglomerate of molecules or particles contributes to providing a multitude of interstitial zones between the liquid sample and the interfacial layer, which considerably increases the amount of surface between these two media and therefore facilitates the fixing of macromolecules or agglomerates of molecules by the dispersed interfacial layer.
  • This fixation is done in almost two-dimensional zones, since it takes place at the level of the interstitial zones, which greatly improves the efficiency and the time of capture of the macromolecules to be concentrated by the interfacial layer.
  • the structure of the functionalized interfacial layer is potentially unstable under the effect of the creation of interfacial area, we can take advantage of Rayleigh instability to produce an emulsion under quasi-static conditions; the densest liquid being above the least dense liquid in the initial conditions. After absorption of the dispersion, we thus find us in the presence of a reformed interfacial layer concentrated in macrolecules or agglomerates of molecules and of a liquid phase corresponding to the liquid sample devoid of all or part of said macromolecules or said agglomerates.
  • the advantage of the present invention is therefore to be able to concentrate quickly and without amplification, the macromolecules or agglomerates and this selectively, passing through the formation of a dispersion which increases the efficiency of the concentration.
  • this step can be carried out in different ways.
  • this dispersion absorption step can be carried out by draining the interstitial films, in the case of a foam, or of the interstitial medium, in the case of an emulsion.
  • the kinetics of resorption can be controlled either by a judicious choice, if necessary, of the molecules constituting the interfacial layer of macromolecules or agglomerates via the length of the molecular chains, for example, using mechanical shearing of the dispersion.
  • the interfacial layer may comprise at least one molecule capable of selectively fixing the macromolecules or the agglomerates. of molecules or particles in question.
  • the molecule capable of fixing the macromolecule or agglomerate of molecules or particles to be concentrated is contained from the start in the interfacial layer before formation of the foam or of the emulsion; it may be a molecule comprising groups capable of fixing the macromolecule or said agglomerate by chemical affinity, electrical or magnetic polarization, and / or ionization, said molecule possibly being preferably a surfactant molecule.
  • said molecule may also be, due to surfactant properties, a dispersion stabilizing molecule formed during one of the process steps.
  • the molecule performs a double function, which is to fix the macromolecule or agglomerate to be concentrated and also to stabilize the dispersion, thus contributing to increase the contact time at the interstitial zones between attractive molecules and macromolecules or agglomerates to be concentrated.
  • the method according to the invention can be applied to the concentration of any type of. macromolecules or agglomerates.
  • nucleic acids As examples, mention may be made, as macromolecules which can be concentrated, according to the method of the invention, of nucleic acids, proteins such as antibodies or antigens.
  • agglomerates of molecules which can be concentrated may be made, according to the process of the invention, prions.
  • agglomerates of particles which can be concentrated colloidal particles such as gold particles.
  • the method according to the invention can be implemented for the concentration of a particular nucleic acid, which is DNA.
  • the interfacial layer corresponds to a second liquid phase comprising a molecule capable of fixing DNA, which is, for example, a molecule functionalized by a probe (such as DNA complementary to DNA to be concentrated) so as to allow specific hybridization of the DNA to be concentrated, for example, a lipid functionalized by a DNA complementary to the DNA to be concentrated.
  • a probe such as DNA complementary to DNA to be concentrated
  • the molecule is a lipid is particularly interesting in the context of this invention, because this category of molecules contributes to to stabilize the dispersion formed.
  • the functionalization of this molecule by a DNA complementary to the DNA to be concentrated allows a concentration and a selective extraction of said DNA.
  • functionalized lipids effective in the concentration of DNA of biotinylated lipids comprising an avidin group or derivative of avidin, onto which the complementary DNA is grafted by a biotinylated end previously incorporated in said Complementary DNA or also cationic lipids comprising at least one spermine group on which the complementary DNA is adsorbed.
  • Such cationic lipids can be lipids of the DOGS or Dioctadecylamidoglycylspermine type, sold under the brand Transfectam TM. These lipids have two saturated carbon chains in C ⁇ 8 as well as a polar head consisting of a spermine group having a strong affinity for DNA. The complementary DNA is thus adsorbed on the spermine sites. The resulting lipids constitute real functionalized probes for specific hybridization of DNA to concentrate, said X DN targets.
  • the interfacial layer corresponds to a second liquid phase deposited on the surface of the liquid sample and comprises vesicles or liposomes composed of phospholipids and containing the antibody or antigen complementary to the antigen or the antibody to be concentrated.
  • the method according to the invention can also be used for the concentration of agglomerates of molecules, such as prions.
  • the interfacial layer corresponds to the interface
  • the interfacial layer being able to selectively concentrate the prions, due to the hydrophobic nature of the prions
  • liquid sample being an aqueous medium such as blood
  • the method according to the invention can also be used for the concentration of colloidal particles.
  • colloidal particles can be, for example, submicron particles of gold solubilized in water (corresponding according to the terminology of the invention to the first liquid phase).
  • the interfacial layer corresponds to a second liquid phase comprising molecules capable of fixing these colloidal gold particles, said molecules being, for example, molecules carrying thiol - SH groups.
  • the concentration process serves, as its name suggests, to selectively concentrate in a interfacial layer a given macromolecule or agglomerate of macromolecules. It can, therefore, be implemented in order to purify, detect or amplify the macromolecule or a given agglomerate of molecules or particles.
  • the subject of the present invention is also a process for purifying a macromolecule or an agglomerate of molecules or particles initially contained in a liquid sample, comprising the concentration of said macromolecule or said agglomerate within said interfacial layer by implementation of the previously described concentration process followed by elimination of the liquid sample depleted in said macromolecule or said agglomerate, after the concentration step.
  • this method finds its application in the case of DNA purification.
  • the method according to the invention consists in starting from molecules functionalized by a specific complementary DNA, in specifically extracting a target DNA, from a liquid sample comprising, for example, a mixture of various DNAs or various portions of DNA, said sample then being eliminated.
  • This process can also find its application in the purification of proteins.
  • the selective capture of proteins via the layer of functionalized lipids, followed by a later stage of crystallization of said proteins can make it possible to isolate these proteins in order to study their structure or even allow to purify a solution of the protein in question.
  • the present invention also relates to a method for detecting a macromolecule or an agglomerate of molecules or particles contained initially in a liquid sample comprising the concentration within an interfacial layer of said macromolecule or of said agglomerate by the implementation of the concentration method described above followed by the detection of said macromolecule or of said agglomerate within said layer by techniques appropriate detection.
  • the detection of DNA when the macromolecule is DNA, the detection of DNA, after selective concentration, can be done by fluorescence excited by laser or by detecting the variation of the electrical surface potential at the level of the functionalized layer, or even by an interfacial rheology technique.
  • the performance of fluorescence or electrical detection can in particular be improved by compressing by a mechanical or hydrodynamic process the interfacial layer containing the hybrid target DNAs at a point on the interface (in the center, for example) coinciding with the laser volume of excitation or with the presence of an electric probe.
  • the present invention also relates to a method for amplifying a macromolecule or an agglomerate of molecules or particles initially contained in a liquid sample comprising the concentration of said macromolecule or said agglomerate within an interfacial layer by the implementation of the concentration process described above, the replacement of said liquid sample, after the step of concentration of said macromolecule or of said agglomerate within said layer, by a liquid comprising amplification agents, followed by the amplification step by means of said agents.
  • the amplification process applies to DNA
  • concentration of the target DNA segments in the interfacial layer the liquid sample depleted in these segments is drawn off and replaced by a purified liquid containing agents amplification such as polymerase and deoxyribonucleotides.
  • the PCR amplification phase can then be carried out without the presence of parasitic DNA segments and the background noise of the PCR is considerably reduced.
  • the amplification method when the amplification method is applied to agglomerates of molecules such as prions, after concentration of the prions in a given interfacial layer, according to the concentration method described above, the liquid sample depleted in said prions can be withdrawn and replaced by a pure liquid containing amplifying agents such as normal proteins PrP not yet transformed. Then, the classic steps of PMCA (sonication, 7) can be implemented without being hindered by parasitic molecules.
  • This amplification process can be followed by ultra-sensitive detection, for example, by fluorescence correlation, carried out jointly or not with PMCA.
  • ultra-sensitive detection for example, by fluorescence correlation
  • we can improve detection performance by concentrating, using a mechanical or hydrodynamic process, the prions locally at a point on the interfacial layer which coincides with the laser measurement volume or with the presence of an electrical probe.
  • FIG. 1 represents the different steps, for arriving at the concentration of a target DNA, according to a particular embodiment of the concentration method according to the invention.
  • FIG. 2 represents a detailed view of an interstitial film created during the formation of a foam.
  • FIGS. 3, 4 and 5 represent purification, detection and concentration methods implemented after the concentration method explained in FIG. 1.
  • FIG. 1 represents, by way of illustration and without limitation, the implementation, in four stages
  • Step (a) of FIG. 1 represents a microbeaker 10, in which is deposited, using a micropipette or syringe, a liquid sample 12 containing the target DNA 14, shown in the figure in the form of strands. Said sample 12 is surmounted by the ambient atmosphere 11.
  • a monolayer 16 comprising a mixture of lipid ligands 18 and lipid diluents 17 (ratio of lipid ligants / lipid diluents 1 : 4) is deposited on the surface of the liquid sample 12.
  • This monolayer 16 corresponds, according to the terminology of the invention, to an interfacial layer.
  • These lipid ligands 18 are such that the target DNAs will be able to hybridize with said lipids. For this, these lipids must, during a preliminary stage, be functionalized.
  • the lipid ligands can be initially biotinylated lipids, of the biotin- (LC) -DPPE type, onto which avidin is first adsorbed and then, in a second step, the DNA complementary to the Target DNA, on avidin by means of a biotin group attached to one of the ends of the complementary DNA.
  • the whole (biotinylated lipid-Avidin-complementary biotinylated DNA) constitutes a lipid functionalized by a probe allowing specific hybridization of the strands of target DNA.
  • the lipids can also be cationic lipids comprising at least one spermine end, on which is adsorbed a DNA complementary to the target DNA to be concentrated. It is understood that the molecules capable of fixing target DNA can extend to any type of molecules capable of selectively fixing the target DNA.
  • a foam-like dispersion 20 is created by injecting air into the liquid sample, said foam consisting of a set of bubbles , maintained in cohesion by interstitial liquid films.
  • the temporary stability of the foam is ensured by the lipids constituting the phase in the form of a monolayer 16.
  • FIG. 2 A detailed view of an interstitial liquid film constituting the foam-type dispersion is shown in FIG. 2.
  • this film has an octahedral shape 22 of very small volume, within which the liquid sample 12 and the monolayer 16 or interfacial layer.
  • the target DNA 14 is almost in direct contact with the functionalized lipids 18 of the monolayer or interfacial layer and is thus very quickly adsorbed by these lipids 18.
  • the fact of passing through a foam multiplies the efficiency and the kinetics of DNA capture by lipids, due to the very small volume of the interstitial films constituting the foam.
  • the foam is resorbed, leaving again a non-dispersed biphasic medium comprising the lipid monolayer 24, of very small thickness, at the level of which adsorbed the target DNA on the hybrid lipids 19, this monolayer corresponding to an interfacial layer referenced 24 in the drawing and a phase 26 corresponding to the liquid sample 12 depleted in target DNA.
  • the lipids 17, 18 and 19 should only be located at the level of the monolayer 16 or interfacial layer 24 (for FIG. 1d), of thickness tiny. However, for reasons of visibility, said lipids have been considerably enlarged.
  • FIGS 3, 4 and 5 illustrate various possible applications of the concentration process, implemented according to a particular embodiment described above.
  • FIG. 3 illustrates the case where, after concentration of said target DNAs, by the method explained in FIG. 1, said DNAs are detected by fluorescence techniques.
  • the lipids 19 functionalized by a DNA complementary to the target DNA and hybrid additionally contain a fluorescent label.
  • the presence of target DNA can be determined at the interfacial layer 24 by a measurement of fluorescence.
  • FIG. 4 illustrates a particular case of purification of a DNA previously concentrated, by the concentration process explained according to FIG. 1.
  • FIG. 5 illustrates the case where the concentration method is used for the sole purpose of obtaining a phase more concentrated in a given DNA than the original phase of said DNA.
  • the layer 24 rich in target DNA is withdrawn using a micropipette 23 to be used for various applications.

Abstract

The invention relates to a method for the selective concentration of a macromolecule or an agglomerate of molecules or particles contained in a liquid sample. The inventive method comprises the following steps, namely: the formation of a foam -or emulsion-type stabilised dispersion from a medium comprising the aforementioned liquid sample and an interfacial layer, whereby said interfacial layer can selectively fix the macromolecule or the agglomerate to be concentrated; and the resorption of the dispersion formed during the preceding step so as to reform said interfacial layer. The invention can be used to concentrate DNA, proteins, prions, colloidal particles.

Description

PROCEDE DE CONCENTRATION DE MACROMOLECULES OU AGGLOMERATS DE MOLECULES OU DE PARTICULES PROCESS FOR THE CONCENTRATION OF MACROMOLECULES OR AGGLOMERATES OF MOLECULES OR PARTICLES
DESCRIPTIONDESCRIPTION
DOMAINE TECHNIQUE La présente invention a trait à un procédé de concentration de macromolëcules ou d'agglomérats de molécules ou de particules, en vue d'une éventuelle détection ou d'une extraction spécifique desdites macromolécules ou desdits agglomérats .TECHNICAL FIELD The present invention relates to a process for the concentration of macromolecules or agglomerates of molecules or particles, with a view to possible detection or specific extraction of said macromolecules or said agglomerates.
ETAT DE LA TECHNIQUE ANTERIEURESTATE OF THE PRIOR ART
Actuellement, les techniques de concentration de macromolécules ou d'agglomérats de molécules sont, pour la plupart, destinées au domaine du diagnostic médical, notamment au domaine de la détection de brins d'ADN ou de complexes protéiniques tels que les antigènes ou les prions.Currently, the techniques for concentrating macromolecules or agglomerates of molecules are, for the most part, intended for the field of medical diagnosis, in particular for the detection of DNA strands or protein complexes such as antigens or prions.
Pour la détection de brins d'ADN, la technique actuelle consiste, le plus souvent, à concentrer des brins d'ADN par amplification dans un milieu liquide, selon la technique communément désignée par l'abréviation PCR (« Polymerase Chain Reaction » ou « Réaction en Chaîne par Polymerase ») .For the detection of DNA strands, the current technique consists, most often, of concentrating DNA strands by amplification in a liquid medium, according to the technique commonly designated by the abbreviation PCR (“Polymerase Chain Reaction” or “ Polymerase Chain Reaction ”).
Cette technique consiste à répliquer les brins d'ADN contenus dans un échantillon liquide un grand nombre de fois (jusqu'à 105-106 fois) en injectant de la polymerase dans l'échantillon soumis à une série de cycles thermiques .This technique consists in replicating the DNA strands contained in a liquid sample a large number of times (up to 10 5 -10 6 times) by injecting polymerase into the sample subjected to a series of thermal cycles.
Après un nombre de cycles thermiques assez important, en tout cas supérieur à 10, la concentration en ADN est suffisamment importante pour permettre la détection.After a fairly large number of thermal cycles, in any case greater than 10, the concentration in DNA is large enough to allow detection.
Toutefois, cette technique, telle qu'explicitée ci-dessus, nécessite une durée importante, du fait du nombre conséquent de cycles thermiques à reproduire pour avoir une quantité d'ADN suffisante. De plus, cette technique s'accompagne d'un bruit de fond important du fait que la polymerase peut amplifier des segments d'ADN présents dans l'échantillon liquide, qui sont de nature différente des segments d'ADN à détecter.However, this technique, as explained above, requires a significant duration, because of the consequent number of thermal cycles to be reproduced in order to have a sufficient quantity of DNA. In addition, this technique is accompanied by a significant background noise because the polymerase can amplify DNA segments present in the liquid sample, which are different in nature from the DNA segments to be detected.
Afin de pallier ces inconvénients, des méthodes alternatives à la PCR ont été développées. Parmi ces méthodes, on peut citer une méthode ne nécessitant pas d'amplification. Le principe de cette méthode de détection sans amplification repose sur la capture des segments d'ADN cibles aussi peu nombreux soient-ils. Elle consiste à hybrider les segments d'ADN cibles avec des nanobilles paramagnétiques fonctionnalisées de manière à concentrer lesdits segments à la surface de ces billes avant détection. Cette méthode se heurte toutefois au problème de l'adsorption non spécifique, certaines billes paramagnétiques, enrobées de latex, se collant aux parois solides du réacteur, au niveau duquel est mise en œuvre la méthode, sous l'effet de l' hydrophobie ou des forces électriques. La sensibilité atteinte n'est alors plus celle escomptée.In order to overcome these drawbacks, alternative methods to PCR have been developed. Among these methods, there may be mentioned a method which does not require amplification. The principle of this detection method without amplification is based on the capture of target DNA segments as small as they are. It consists in hybridizing the target DNA segments with functionalized paramagnetic nanobeads so as to concentrate said segments on the surface of these beads before detection. This method, however, faces the problem of non-specific adsorption, certain paramagnetic beads, coated with latex, sticking to the solid walls of the reactor, at the level of which the method is implemented, under the effect of hydrophobicity or electrical forces. The sensitivity reached is then no longer that expected.
En ce qui concerne la détection de protéines telles que les anticorps ou les antigênes, deux types de tests se distinguent : les tests dits « en phase homogène » et ceux en « phase hétérogène » . Les tests en phase homogène ont lieu en solution. La mesure de la quantité d'anticorps est réalisée directement dans la solution par fixation de cet anticorps sur l'antigène complémentaire, sans qu'il y ait de séparation physique entre les anticorps libres et les anticorps liés à un antigène. Cette distinction entre anticorps libre et anticorps lié est faite grâce à diverses astuces, notamment basées, par exemple, sur un transfert de fluorescence entre deux anticorpsWith regard to the detection of proteins such as antibodies or antigens, two types of tests are distinguished: the so-called tests "In homogeneous phase" and those in "heterogeneous phase". Homogeneous phase tests take place in solution. The amount of antibody is measured directly in the solution by fixing this antibody to the complementary antigen, without there being any physical separation between the free antibodies and the antibodies bound to an antigen. This distinction between free antibody and bound antibody is made thanks to various tricks, notably based, for example, on a fluorescence transfer between two antibodies
(l'antigène étant pris en sandwich entre deux anticorps marqués par exemple). Ces tests présentent l'avantage d'être rapides, et peu coûteux. Ce sont eux qui sont massivement utilisés dans les programmes de criblage haut débit de l'industrie pharmaceutique par exemple. Toutefois, leur sensibilité est limitée, aux environs de lnM en ce qui concerne les tests basés sur les acides nucléiques. C'est pourquoi l'industrie du diagnostic utilise préfërentiellement des tests en phase hétérogène, dans lesquels un anticorps est immobilisé sur un support solide, support qu'il sera alors possible de laver pour éliminer la quasi-totalité des réactifs de marquage. De plus, une amplification du signal est réalisée, en utilisant une enzyme comme molécule de marquage. Ces tests sur support solide sont plus lents et plus onéreux que ceux en phase homogène, mais ils permettent d'atteindre des sensibilités bien plus grandes, aux alentours de 0,1 pM, soit 10 000 fois mieux que les tests en phase homogène. Toutefois, l'utilisation d'un support solide rend ces tests difficiles à mettre en œuvre. En ce qui concerne les complexes protéiniques tels que les prions, ceux-ci doivent également, pour être détectables, dans des liquides physiologiques, tels que le sang, subir une phase de concentration. Pour ces molécules, la technique PCR précédemment décrite est inutilisable, parce qu'ils ne contiennent pas de nucléotides. De ce fait, des recherches relatives au domaine des prions ont abouti récemment à la mise en place d'une méthode dite λméthode d'amplification des protéines anormalement repliées' ou PMCA (pour xProtein Misfolding Cyclic Amplification' ) .(the antigen being sandwiched between two labeled antibodies for example). These tests have the advantage of being rapid and inexpensive. They are the ones that are used extensively in broadband screening programs in the pharmaceutical industry, for example. However, their sensitivity is limited, around 1 nM with regard to tests based on nucleic acids. This is why the diagnostic industry preferably uses tests in heterogeneous phase, in which an antibody is immobilized on a solid support, support which it will then be possible to wash in order to remove almost all of the labeling reagents. In addition, an amplification of the signal is carried out, using an enzyme as a labeling molecule. These tests on solid support are slower and more expensive than those in homogeneous phase, but they make it possible to reach much greater sensitivities, around 0.1 pM, or 10,000 times better than tests in homogeneous phase. However, the use of a solid support makes these tests difficult to implement. Regarding protein complexes such as prions, these must also, in order to be detectable, in physiological fluids, such as blood, undergo a concentration phase. For these molecules, the PCR technique described above cannot be used, because they do not contain nucleotides. As a result, research relating to the prion domain has recently resulted in the establishment of a method called λ abnormally folded protein amplification method 'or PMCA (for x Protein Misfolding Cyclic Amplification').
Les prions, responsables notamment des encéphalopathies spongiformes, sont des complexes ou agglomérats constitués d'une protéine naturelle, la glycoprotéine PrPc, normalement présente à la surface de nombreuses cellules dans l'organisme et d'une protéine infectieuse PrPsc, qui ne se différencie de la glycoprotéine normale PrPc que par sa conformation, qui est anormalement repliée. Les protéines PrPsc sont aptes, d'une part, à s'associer aux protéines PrPc et d'autre part, aptes à induire la transformation de protéines normales en protéines infectieuses. La détection des prions est rendue difficile par le fait qu'ils ne sont présents en quantité notable que dans le cerveau, alors qu'ils se retrouvent à l'état de traces dans le sang.Prions, responsible in particular for spongiform encephalopathies, are complexes or agglomerates made up of a natural protein, the glycoprotein PrP c , normally present on the surface of many cells in the body and of an infectious protein PrP sc , which does not differs from normal PrP c glycoprotein only by its conformation, which is abnormally folded. PrP sc proteins are able, on the one hand, to associate with PrP c proteins and on the other hand, able to induce the transformation of normal proteins into infectious proteins. Detection of prions is made difficult by the fact that they are present in notable quantities only in the brain, whereas they are found in traces in the blood.
La technique PMCA permet ainsi de concentrer les prions dans le milieu de prélèvement, c'est-à-dire le sang, afin qu'ils puissent être détectés . Pour ce faire, un procédé de sonication destiné à fragmenter les prions est mis en œuvre. Tous les prions issus de cette fragmentation par ultra-sons, repoussent in-vitro à l'aide des protéines PrPc de l'échantillon. Ce cycle élémentaire (fragmentation- repousse) est reproduit autant de fois que nécessaire, jusqu'à ce que la quantité de prions devienne détectable.The PMCA technique thus makes it possible to concentrate the prions in the sampling medium, that is to say the blood, so that they can be detected. To do this, a sonication process intended to fragment the prions is implemented. All the prions resulting from this ultrasonic fragmentation regrow in-vitro using the PrP c proteins in the sample. This elementary cycle (fragmentation-regrowth) is repeated as many times as necessary, until the quantity of prions becomes detectable.
L'étape de détection, facilitée par l'étape d'amplification, est effectuée par spectroscopie de fluorescence. Les protéines à détecter sont marquées à l'aide d'une sonde fluorescente, dont la présence se manifeste lorsque celle-ci est éclairée par une lumière de longueur d'onde caractéristique. Toutefois, cette technique engendre, le plus souvent, un bruit de fond non négligeable, du fait que les sondes peuvent s'associer à d'autres molécules que les prions à détecter.The detection step, facilitated by the amplification step, is carried out by fluorescence spectroscopy. The proteins to be detected are labeled using a fluorescent probe, the presence of which is manifested when this is illuminated by light of characteristic wavelength. However, this technique generally generates a significant background noise, since the probes can associate with other molecules than the prions to be detected.
Une technique plus élaborée, dite technique de corrélation de fluorescence, met en œuvre deux sondes fluorescentes qui émettent selon deux longueurs d'onde différentes et qui s'accrochent toutes deux symétriquement et spécifiquement sur les protéines PrPc, ces dernières se trouvant en concentration importante au sein des prions. On éclaire ensuite avec des faisceaux à deux longueurs d'onde différentes, un volume très petit du liquide contenant les prions à détecter et on détecte, de manière instationnaire, les deux émissions fluorescentes intenses dues à la présence de prions, qui constituent un agglomérat de protéines PrP normales ou infectieuses sur lesquelles sont accrochées les sondes fluorescentes. Toutefois, un bruit de fond subsiste, dû notamment à la présence de sondes fluorescentes n'ayant pu trouver de sites d'adsorption spécifiques et à la présence de protéines normales PrPc isolées, auxquelles se sont accrochées des sondes fluorescentes.A more elaborate technique, known as the fluorescence correlation technique, uses two fluorescent probes which emit at two different wavelengths and which both hang symmetrically and specifically on PrP c proteins, the latter being in significant concentration within prions. A very small volume of the liquid containing the prions to be detected is then illuminated with beams at two different wavelengths, and the two intense fluorescent emissions due to the presence of prions, which constitute an agglomerate of normal or infectious PrP proteins on which the fluorescent probes are attached. However, background noise remains, due in particular to the presence of fluorescent probes which could not find specific adsorption sites and to the presence of normal PrP c proteins isolated, to which fluorescent probes have become attached.
Ainsi, les procédés de concentration, exposés précédemment, présentent tous l'un ou plusieurs des inconvénients suivants :Thus, the concentration methods, described above, all have one or more of the following drawbacks:
-ils ne permettent pas une concentration spécifique des macromolécules ou agglomérats à concentrer, du fait que certaines de ces techniques peuvent générer des espèces, pouvant entraîner, notamment lors d'une éventuelle détection, un bruit de fond important ;-they do not allow a specific concentration of the macromolecules or agglomerates to be concentrated, because some of these techniques can generate species, which can cause, notably during a possible detection, a significant background noise;
-ils ne permettent pas une concentration suffisante des macromolécules ou agglomérats en vue d'une éventuelle détection.-they do not allow a sufficient concentration of macromolecules or agglomerates for possible detection.
EXPOSE DE L'INVENTION.PRESENTATION OF THE INVENTION.
La présente invention a précisément pour objet de proposer un procédé de concentration de macrolecules ou d'agglomérats de molécules ou de particules, qui permette notamment une concentration sélective desdites macromolécules ou desdits agglomérats, en vue d'une éventuelle détection en limitant autant que possible les bruits de fond, ou en vue d'une éventuelle purification d'un échantillon contenant lesdites macromolécules ou lesdits agglomérats .The object of the present invention is precisely to propose a process for the concentration of macrolecules or agglomerates of molecules or particles, which allows in particular a selective concentration of said macromolecules or said agglomerates, with a view to possible detection by limiting as much as possible the background noise, or for possible purification of a sample containing said macromolecules or said agglomerates.
Pour ce faire, la présente invention a pour objet un procédé de concentration sélective d'une macromolècule ou d'un agglomérat de molécules ou de particules contenu dans un échantillon liquide, comprenant successivement les étapes suivantes :To do this, the subject of the present invention is a process for the selective concentration of a macromolecule or an agglomerate of molecules or particles contained in a liquid sample, successively comprising the following steps:
- formation d'une dispersion stabilisée de type mousse ou émulsion, à partir d'un milieu comprenant ledit échantillon liquide et une couche interfaciale, ladite couche interfaciale étant apte à fixer sélectivement ladite macromolécule ou ledit agglomérat à concentrer ; et - résorption de la dispersion formée lors de l'étape précédente de façon à reformer ladite couche interfaciale.- Formation of a stabilized dispersion of foam or emulsion type, from a medium comprising said liquid sample and an interfacial layer, said interfacial layer being capable of selectively fixing said macromolecule or said agglomerate to be concentrated; and - resorption of the dispersion formed during the previous step so as to reform said interfacial layer.
Par couche interfaciale, on entend, selon l'invention, une monocouche (ou zone quasi- bidimensionnelle) localisée à la surface de l'échantillon liquide (dite première phase liquide) comprenant la macromolécule ou l'agglomérat à concentrer. De part sa nature et ses propriétés spécifiques, cette couche est apte à assurer le transfert sélectif de la macromolécule ou de l'agglomérat de l'échantillon liquide vers la couche interfaciale, et, du fait de son volume infime par rapport à l'échantillon liquide, à concentrer ladite macromolécule ou ledit agglomérat . Selon la nature de la macromolècule ou de l'agglomérat à concentrer, la couche interfaciale peut correspondre à une deuxième phase liquide déposée à la surface de l'échantillon liquide (cas par exemple, lorsqu'il s'agit de concentrer une macromolècule du type ADN) , cette phase présentant des caractéristiques telles qu'elle permet d'attirer la macromolècule ou l'agglomérat vers elle. La couche interfaciale peut correspondre également à l'interface entre l'atmosphère ambiante et l'échantillon liquide (par exemple, lorsque la macromolècule ou l'agglomérat à concentrer présente un caractère hydrophobe, tel que les prions, et que l'échantillon liquide le contenant est un milieu aqueux) .By interfacial layer is meant, according to the invention, a monolayer (or almost two-dimensional area) located on the surface of the liquid sample (called first liquid phase) comprising the macromolecule or the agglomerate to be concentrated. Due to its nature and specific properties, this layer is capable of ensuring the selective transfer of the macromolecule or of the agglomerate from the liquid sample to the interfacial layer, and, because of its minute volume compared to the sample liquid, to concentrate said macromolecule or said agglomerate. Depending on the nature of the macromolecule or the agglomerate to be concentrated, the interfacial layer may correspond to a second liquid phase deposited on the surface of the liquid sample (case for example, when it is a question of concentrating a macromolecule of the DNA type), this phase having characteristics such that it makes it possible to attract the macromolecule or the agglomerate towards it. The interfacial layer can also correspond to the interface between the ambient atmosphere and the liquid sample (for example, when the macromolecule or the agglomerate to be concentrated has a hydrophobic character, such as prions, and when the liquid sample containing is an aqueous medium).
Dans le cas où la couche interfaciale correspond à une deuxième phase liquide, le procédé de l'invention peut comprendre une étape préalable (située avant l'étape de formation de la dispersion) consistant à déposer à la surface de l'échantillon liquide ladite deuxième phase liquide ou couche interfaciale.In the case where the interfacial layer corresponds to a second liquid phase, the method of the invention may comprise a prior step (located before the step of forming the dispersion) consisting in depositing said second on the surface of the liquid sample liquid phase or interfacial layer.
Généralement, l'étape de formation de la dispersion est effectuée par agitation mécanique du milieu comprenant l'échantillon liquide et la couche interfaciale ou par injection directement dans l'échantillon liquide surmontée de la couche interfaciale de jets capillaires gazeux ou liquides.Generally, the step of forming the dispersion is carried out by mechanical agitation of the medium comprising the liquid sample and the interfacial layer or by injection directly into the liquid sample surmounted by the interfacial layer of gaseous or liquid capillary jets.
On note qu'une mousse désigne une dispersion comprenant un ensemble de bulles de gaz (typiquement d'air) coexistant avec un milieu liquide interstitiel, sous forme de minces films intersticiels entre les bulles. Ce type de dispersion ménage ainsi une multitude .d'interfaces liquide-gaz. La mousse, selon l'invention, peut être obtenue, par exemple, par agitation mécanique vigoureuse du milieu susmentionné ou par injection au sein de ce milieu de jets capillaires gazeux (typiquement des jets d'air). On note que, selon l'invention, une emulsion désigne une dispersion, dans laquelle la couche interfaciale est divisée en globules au sein de l'échantillon liquide, ledit échantillon liquide constituant un milieu interstitiel. On forme ainsi une multitude d'interfaces liquide-liquide, et on augmente de ce fait considérablement la surface de contact entre l'échantillon liquide et la couche interfaciale.It is noted that a foam designates a dispersion comprising a set of gas bubbles (typically air) coexisting with an interstitial liquid medium, in the form of thin interstitial films between the bubbles. This type of dispersion thus provides a multitude of liquid-gas interfaces. The foam, according to the invention can be obtained, for example, by vigorous mechanical agitation of the abovementioned medium or by injection into this medium of gaseous capillary jets (typically air jets). It should be noted that, according to the invention, an emulsion designates a dispersion, in which the interfacial layer is divided into globules within the liquid sample, said liquid sample constituting an interstitial medium. A multitude of liquid-liquid interfaces is thus formed, and the contact surface between the liquid sample and the interfacial layer is considerably increased.
L' emulsion, au même titre que la mousse, peut être obtenue par agitation mécanique du milieu comprenant l'échantillon liquide et la couche interfaciale mais également par injection directe dans l'échantillon liquide surmonté de la couche interfaciale de jets capillaires liquides ou gazeux.The emulsion, in the same way as the foam, can be obtained by mechanical agitation of the medium comprising the liquid sample and the interfacial layer but also by direct injection into the liquid sample surmounted by the interfacial layer of liquid or gaseous capillary jets.
Ainsi, selon l'invention, le fait de passer par une dispersion du type mousse ou emulsion pour assurer la concentration d'une macromolécule ou d'un agglomérat de molécules ou particules contribue à ménager une multitude de zones interstitielles entre l'échantillon liquide et la couche interfaciale, ce qui augmente considérablement la quantité de surface entre ces deux milieux et facilite, de ce fait, la fixation des macromolécules ou les agglomérats de molécules par la couche interfaciale dispersée. Cette fixation se fait dans des zones quasi- bidimensionnelles, du fait qu'elle a lieu au niveau des zones interstitielles, ce qui améliore grandement l'efficacité et le temps de capture des macromolécules à concentrer par la couche interfaciale.Thus, according to the invention, the fact of going through a dispersion of the foam or emulsion type to ensure the concentration of a macromolecule or of an agglomerate of molecules or particles contributes to providing a multitude of interstitial zones between the liquid sample and the interfacial layer, which considerably increases the amount of surface between these two media and therefore facilitates the fixing of macromolecules or agglomerates of molecules by the dispersed interfacial layer. This fixation is done in almost two-dimensional zones, since it takes place at the level of the interstitial zones, which greatly improves the efficiency and the time of capture of the macromolecules to be concentrated by the interfacial layer.
Si la structure de la couche interfaciale fonctionnalisée est potentiellement instable sous l'effet de la création d'aire interfaciale, on pourra mettre à profit l'instabilité de Rayleigh pour réaliser une emulsion dans des conditions quasi-statiques ; le liquide le plus dense se trouvant au-dessus du liquide le moins dense dans les conditions initiales. Après résorption de la dispersion, on se retrouve, ainsi, en présence d'une couche interfaciale reformée concentrée en macrolecules ou agglomérats de molécules et d'une phase liquide correspondant à l'échantillon liquide dépourvu en tout ou partie desdites macromolêcules ou desdits agglomérats .If the structure of the functionalized interfacial layer is potentially unstable under the effect of the creation of interfacial area, we can take advantage of Rayleigh instability to produce an emulsion under quasi-static conditions; the densest liquid being above the least dense liquid in the initial conditions. After absorption of the dispersion, we thus find ourselves in the presence of a reformed interfacial layer concentrated in macrolecules or agglomerates of molecules and of a liquid phase corresponding to the liquid sample devoid of all or part of said macromolecules or said agglomerates.
L'avantage de la présente invention est donc de pouvoir concentrer rapidement et sans amplification, les macromolécules ou agglomérats et ceci de manière sélective, en passant par la formation d'une dispersion qui augmente l'efficacité de la concentration.The advantage of the present invention is therefore to be able to concentrate quickly and without amplification, the macromolecules or agglomerates and this selectively, passing through the formation of a dispersion which increases the efficiency of the concentration.
Comme décrit précédemment, après capture des macromolécules ou agglomérat de molécules ou particules à concentrer, la dispersion est amenée à subir une étape de résorption. Cette étape peut être menée de différentes manières. Par exemple, cette étape de résorption de la dispersion peut être effectuée par drainage des films interstitiels, dans le cas d'une mousse, ou du milieu interstitiel, dans le cas d'une emulsion. La cinétique de la résorption peut être contrôlée soit par un choix judicieux, le cas échéant, des molécules constitutives de la couche interfaciale des macromolécules ou agglomérats via la longueur des chaînes moléculaires, par exemple, à l'aide d'un cisaillement mécanique de la dispersion.As described above, after capture of the macromolecules or agglomerate of molecules or particles to be concentrated, the dispersion is brought to undergo a resorption step. This step can be carried out in different ways. For example, this dispersion absorption step can be carried out by draining the interstitial films, in the case of a foam, or of the interstitial medium, in the case of an emulsion. The kinetics of resorption can be controlled either by a judicious choice, if necessary, of the molecules constituting the interfacial layer of macromolecules or agglomerates via the length of the molecular chains, for example, using mechanical shearing of the dispersion.
Selon la nature de la couche interfaciale et de la macromolécule ou de l'agglomérat à concentrer, et notamment lorsque le couche interfaciale correspond à une deuxième phase liquide, la couche interfaciale peut comprendre au moins une molécule apte à fixer sélectivement les macromolécules ou les agglomérats de molécules ou particules en question.Depending on the nature of the interfacial layer and of the macromolecule or of the agglomerate to be concentrated, and in particular when the interfacial layer corresponds to a second liquid phase, the interfacial layer may comprise at least one molecule capable of selectively fixing the macromolecules or the agglomerates. of molecules or particles in question.
Selon ce mode de réalisation, la molécule apte à fixer la macromolécule ou agglomérat de molécules ou particules à concentrer est contenue dès le départ dans la couche interfaciale avant formation de la mousse ou de l' emulsion ; elle peut être une molécule comportant des groupements aptes à fixer la macromolécule ou ledit agglomérat par affinité chimique, polarisation électrique ou magnétique, et/ou ionisation, ladite molécule pouvant être, de préférence, une molécule surfactante.According to this embodiment, the molecule capable of fixing the macromolecule or agglomerate of molecules or particles to be concentrated is contained from the start in the interfacial layer before formation of the foam or of the emulsion; it may be a molecule comprising groups capable of fixing the macromolecule or said agglomerate by chemical affinity, electrical or magnetic polarization, and / or ionization, said molecule possibly being preferably a surfactant molecule.
Si ladite molécule n'est pas mélangée avec d'autres molécules surfactantes, ladite molécule peut être également, du fait de propriétés surfactantes, une molécule stabilisatrice de la dispersion formée au cours d'une des étapes du procédé.If said molecule is not mixed with other surfactant molecules, said molecule may also be, due to surfactant properties, a dispersion stabilizing molecule formed during one of the process steps.
Dans ce cas, la molécule assure une double fonction, qui est de fixer la macromolécule ou l'agglomérat à concentrer et également d'assurer la stabilisation de la dispersion, contribuant ainsi à augmenter le temps de contact au niveau des zones interstitielles entre molécules attractives et macromolécules ou agglomérats à concentrer.In this case, the molecule performs a double function, which is to fix the macromolecule or agglomerate to be concentrated and also to stabilize the dispersion, thus contributing to increase the contact time at the interstitial zones between attractive molecules and macromolecules or agglomerates to be concentrated.
Le procédé selon l'invention peut s'appliquer à la concentration de tout type de. macromolécules ou d'agglomérats.The method according to the invention can be applied to the concentration of any type of. macromolecules or agglomerates.
A titre d'exemples, on peut citer comme macromolécules pouvant être concentrées, selon le procédé de l'invention, les acides nucléiques, les protéines telles les anticorps ou les antigènes.As examples, mention may be made, as macromolecules which can be concentrated, according to the method of the invention, of nucleic acids, proteins such as antibodies or antigens.
A titre d'exemples, on peut citer comme agglomérats de molécules pouvant être concentrés, selon le procédé de l'invention, les prions. A titre d'exemples, on peut citer comme agglomérats de particules pouvant être concentrés des particules colloïdales telles que des particules d'or.Mention may be made, as examples, of agglomerates of molecules which can be concentrated, according to the process of the invention, prions. By way of examples, there may be mentioned as agglomerates of particles which can be concentrated colloidal particles such as gold particles.
Ainsi, le procédé selon l'invention peut être mis en œuvre pour la concentration d'un acide nucléique particulier, qui est l'ADN.Thus, the method according to the invention can be implemented for the concentration of a particular nucleic acid, which is DNA.
Dans le cas de l'ADN, la couche interfaciale correspond à une deuxième phase liquide comprenant une molécule apte à fixer l'ADN, qui est, par exemple, une molécule fonctionnalisée par une sonde (telle qu'un ADN complémentaire de l'ADN à concentrer) de façon à permettre l'hybridation spécifique de l'ADN à concentrer, par exemple, un lipide fonctionnalisé par un ADN complémentaire de l'ADN à concentrer.In the case of DNA, the interfacial layer corresponds to a second liquid phase comprising a molecule capable of fixing DNA, which is, for example, a molecule functionalized by a probe (such as DNA complementary to DNA to be concentrated) so as to allow specific hybridization of the DNA to be concentrated, for example, a lipid functionalized by a DNA complementary to the DNA to be concentrated.
Le fait que la molécule soit un lipide est particulièrement intéressant dans le cadre de cette invention, car cette catégorie de molécules contribue à assurer la stabilisation de la dispersion formée. De plus, la fonctionnalisation de cette molécule par un ADN complémentaire de l'ADN à concentrer permet une concentration et une extraction sélective dudit ADN. A titre d'exemples, on peut citer, comme lipides fonctionnalisés efficaces dans la concentration d'ADN, des lipides biotinylés comportant un groupement avidine ou dérivé de l'avidine, sur lequel est greffé l'ADN complémentaire par une extrémité biotinylée précédemment incorporée audit ADN complémentaire ou encore des lipides cationiques comprenant au moins un groupement spermine sur lequel est adsorbé l'ADN complémentaire. De tels lipides cationiques peuvent être des lipides du type DOGS ou Dioctadécylamidoglycylspermine, commercialisé sous la marque Transfectam™. Ces lipides présentent deux chaînes carbonées saturées en Cι8 ainsi qu'une tête polaire constituée d'un groupement spermine présentant une forte affinité pour l'ADN. L'ADN complémentaire est ainsi adsorbé sur les sites spermine. Les lipides résultant constituent de véritables sondes fonctionnalisées permettant l'hydridation spécifique des ADN à concentrer, dit X DN cibles' .The fact that the molecule is a lipid is particularly interesting in the context of this invention, because this category of molecules contributes to to stabilize the dispersion formed. In addition, the functionalization of this molecule by a DNA complementary to the DNA to be concentrated allows a concentration and a selective extraction of said DNA. By way of examples, mention may be made, as functionalized lipids effective in the concentration of DNA, of biotinylated lipids comprising an avidin group or derivative of avidin, onto which the complementary DNA is grafted by a biotinylated end previously incorporated in said Complementary DNA or also cationic lipids comprising at least one spermine group on which the complementary DNA is adsorbed. Such cationic lipids can be lipids of the DOGS or Dioctadecylamidoglycylspermine type, sold under the brand Transfectam ™. These lipids have two saturated carbon chains in Cι 8 as well as a polar head consisting of a spermine group having a strong affinity for DNA. The complementary DNA is thus adsorbed on the spermine sites. The resulting lipids constitute real functionalized probes for specific hybridization of DNA to concentrate, said X DN targets.
Le procédé selon l'invention peut être utilisé pour la concentration sélective d'antigènes ou d'anticorps contenus dans un échantillon liquide, sans passer par un support solide, tel que cela est le cas des réalisations de l'art antérieur. Selon ce mode de réalisation, la couche interfaciale correspond à une deuxième phase liquide déposée à la surface de l'échantillon liquide et comprend des vésicules ou liposomes constitués de phospholipides et contenant l'anticorps ou l'antigène complémentaire de l'antigène ou l'anticorps à concentrer.The method according to the invention can be used for the selective concentration of antigens or antibodies contained in a liquid sample, without passing through a solid support, as is the case with the embodiments of the prior art. According to this embodiment, the interfacial layer corresponds to a second liquid phase deposited on the surface of the liquid sample and comprises vesicles or liposomes composed of phospholipids and containing the antibody or antigen complementary to the antigen or the antibody to be concentrated.
Le procédé selon l'invention peut également être utilisé pour la concentration d'agglomérats de molécules, tels que les prions. Dans le cas des prions, la couche interfaciale correspond à l'interfaceThe method according to the invention can also be used for the concentration of agglomerates of molecules, such as prions. In the case of prions, the interfacial layer corresponds to the interface
(échantillon liquide/atmosphère ambiante) , la couche interfaciale étant apte à concentrer sélectivement les prions, du fait du caractère hydrophobe des prions(liquid sample / ambient atmosphere), the interfacial layer being able to selectively concentrate the prions, due to the hydrophobic nature of the prions
(l'échantillon liquide étant un milieu aqueux tel que le. sang) .(the liquid sample being an aqueous medium such as blood).
Le procédé selon l'invention peut également être utilisé pour la concentration de particules colloïdales. Celles-ci peuvent être, par exemple, des particules submicroniques d'or solubilisées dans l'eau (correspondant selon la terminologie de l'invention à la première phase liquide) . Dans ce cas, la couche interfaciale correspond à une deuxième phase liquide comprenant des molécules aptes à fixer ces particules colloïdales d'or, lesdites molécules étant, par exemple, des molécules portant des groupements thiol - SH.The method according to the invention can also be used for the concentration of colloidal particles. These can be, for example, submicron particles of gold solubilized in water (corresponding according to the terminology of the invention to the first liquid phase). In this case, the interfacial layer corresponds to a second liquid phase comprising molecules capable of fixing these colloidal gold particles, said molecules being, for example, molecules carrying thiol - SH groups.
Le procédé de concentration, explicité ci- dessus, sert, comme son nom l'indique, à concentrer sélectivement dans une couche interfaciale une macromolécule ou un agglomérat de macromolécules donné. Il peut, de ce fait, être mis en œuvre, afin de purifier, de détecter ou d'amplifier la macromolécule ou un agglomérat de molécules ou particules donné. Ainsi, la présente invention a également pour objet un procédé de purification d'une macromolécule ou d'un agglomérat de molécules ou particules contenu initialement dans un échantillon liquide, comprenant la concentration de ladite macromolècule ou dudit agglomérat au sein de ladite couche interfaciale par la mise en oeuvre du procédé de concentration décrit précédemment suivie de l'élimination de l'échantillon liquide appauvri en ladite macromolècule ou ledit agglomérat, après l'étape de concentration.The concentration process, explained above, serves, as its name suggests, to selectively concentrate in a interfacial layer a given macromolecule or agglomerate of macromolecules. It can, therefore, be implemented in order to purify, detect or amplify the macromolecule or a given agglomerate of molecules or particles. Thus, the subject of the present invention is also a process for purifying a macromolecule or an agglomerate of molecules or particles initially contained in a liquid sample, comprising the concentration of said macromolecule or said agglomerate within said interfacial layer by implementation of the previously described concentration process followed by elimination of the liquid sample depleted in said macromolecule or said agglomerate, after the concentration step.
Par exemple, ce procédé trouve son application dans le cas de la purification d'ADN. Dans ce cas, le procédé selon l'invention consiste à partir de molécules fonctionnalisées par un ADN complémentaire spécifique, à extraire spécifiquement un ADN cible, à partir d'un échantillon liquide comprenant, par exemple, un mélange de divers ADN ou diverses portions d'ADN, ledit échantillon étant ensuite éliminé. Ce procédé peut également trouver son application dans la purification de protéines. La capture sélective de protéines via la couche de lipides fonctionnalisés, suivi d'une étape ultérieure de cristallisation desdites protéines peut permettre d'isoler ces protéines afin d'en étudier leur structure ou bien permettre de purifier une solution de la protéine en question.For example, this method finds its application in the case of DNA purification. In this case, the method according to the invention consists in starting from molecules functionalized by a specific complementary DNA, in specifically extracting a target DNA, from a liquid sample comprising, for example, a mixture of various DNAs or various portions of DNA, said sample then being eliminated. This process can also find its application in the purification of proteins. The selective capture of proteins via the layer of functionalized lipids, followed by a later stage of crystallization of said proteins can make it possible to isolate these proteins in order to study their structure or even allow to purify a solution of the protein in question.
La présente invention a également pour objet un procédé de détection d'une macromolécule ou d'un agglomérat de molécules ou particules contenu initialement dans un échantillon liquide comprenant la concentration au sein d'une couche interfaciale de ladite macromolécule ou dudit agglomérérat par la mise en oeuvre du procédé de concentration décrit précédemment suivie de la détection de ladite macromolécule ou dudit agglomérat au sein de ladite couche par des techniques appropriées de détection.The present invention also relates to a method for detecting a macromolecule or an agglomerate of molecules or particles contained initially in a liquid sample comprising the concentration within an interfacial layer of said macromolecule or of said agglomerate by the implementation of the concentration method described above followed by the detection of said macromolecule or of said agglomerate within said layer by techniques appropriate detection.
Ainsi, lorsque la macromolécule est de l'ADN, la détection de l'ADN, après concentration sélective, peut se faire par fluorescence excitée par laser ou en détectant la variation du potentiel électrique de surface au niveau de la couche fonctionnalisée, ou encore par une -technique de rhéologie interfaciale. Les performances de la détection par fluorescence ou électrique peuvent être notamment améliorées en comprimant par un procédé mécanique ou hydrodynamique la couche interfaciale contenant les ADN cibles hybrides en un point de l'interface (au centre, par exemple) coïncidant avec le volume laser d'excitation ou bien avec la présence d'une sonde électrique.Thus, when the macromolecule is DNA, the detection of DNA, after selective concentration, can be done by fluorescence excited by laser or by detecting the variation of the electrical surface potential at the level of the functionalized layer, or even by an interfacial rheology technique. The performance of fluorescence or electrical detection can in particular be improved by compressing by a mechanical or hydrodynamic process the interfacial layer containing the hybrid target DNAs at a point on the interface (in the center, for example) coinciding with the laser volume of excitation or with the presence of an electric probe.
Enfin, la présente invention a également pour objet un procédé d'amplification d'une macromolécule ou d'un agglomérat de molécules ou particules contenu initialement dans un échantillon liquide comprenant la concentration de ladite macromolécule ou dudit agglomérat au sein d'une couche interfaciale par la mise en oeuvre du procédé de concentration décrit précédemment, le remplacement dudit échantillon liquide, après l'étape de concentration de ladite macromolécule ou dudit agglomérat au sein de ladite couche, par un liquide comprenant des agents d'amplification, suivie de l'étape d'amplification au moyen desdits agents. Ainsi, lorsque le procédé d'amplification s'applique à l'ADN, après concentration des segments d'ADN cibles dans la couche interfaciale, l'échantillon liquide appauvri en ces segments est soutiré et remplacé par un liquide purifié contenant des agents d'amplification tels que la polymerase et des désoxyribonucléotides . La phase d'amplification par PCR peut être alors réalisée sans la présence de segments d'ADN parasites et le bruit de fond de la PCR s'en trouve considérablement réduit.Finally, the present invention also relates to a method for amplifying a macromolecule or an agglomerate of molecules or particles initially contained in a liquid sample comprising the concentration of said macromolecule or said agglomerate within an interfacial layer by the implementation of the concentration process described above, the replacement of said liquid sample, after the step of concentration of said macromolecule or of said agglomerate within said layer, by a liquid comprising amplification agents, followed by the amplification step by means of said agents. Thus, when the amplification process applies to DNA, after concentration of the target DNA segments in the interfacial layer, the liquid sample depleted in these segments is drawn off and replaced by a purified liquid containing agents amplification such as polymerase and deoxyribonucleotides. The PCR amplification phase can then be carried out without the presence of parasitic DNA segments and the background noise of the PCR is considerably reduced.
Par exemple, lorsque le procédé d'amplification s'applique à des agglomérats de molécules tels que les prions, après concentration des prions dans une couche interfaciale donnée, selon le procédé de concentration décrit précédemment, l'échantillon liquide appauvrie en lesdits prions peut être soutirée et remplacée par un liquide pur contenant des agents d'amplification tels que des protéines normales PrP non encore transformées. Ensuite, les étapes classiques de la PMCA (sonication, ...) peuvent être mises en œuvre sans être gênées par des molécules parasites .For example, when the amplification method is applied to agglomerates of molecules such as prions, after concentration of the prions in a given interfacial layer, according to the concentration method described above, the liquid sample depleted in said prions can be withdrawn and replaced by a pure liquid containing amplifying agents such as normal proteins PrP not yet transformed. Then, the classic steps of PMCA (sonication, ...) can be implemented without being hindered by parasitic molecules.
Ce procédé d'amplification peut être suivi d'une détection ultra-sensible, par exemple, par corrélation de fluorescence, effectuée conjointement ou non à la PMCA. Par cette technique, on peut améliorer les performances de la détection en concentrant, à l'aide d'un procédé mécanique ou hydrodynamique, les prions localement en un point de la couche interfaciale qui coïncide avec le volume de mesure laser ou bien avec la présence d'une sonde électrique.This amplification process can be followed by ultra-sensitive detection, for example, by fluorescence correlation, carried out jointly or not with PMCA. By this technique, we can improve detection performance by concentrating, using a mechanical or hydrodynamic process, the prions locally at a point on the interfacial layer which coincides with the laser measurement volume or with the presence of an electrical probe.
D'autres caractéristiques et avantages de l'invention apparaîtront mieux à la lecture de la description du mode de réalisation particulier qui suit, en référence aux dessins annexés.Other characteristics and advantages of the invention will appear better on reading the description of the particular embodiment which follows, with reference to the accompanying drawings.
BREVE DESCRIPTION DES FIGURES.BRIEF DESCRIPTION OF THE FIGURES.
La figure 1 représente les différentes étapes, pour arriver à la concentration d'un ADN cible, selon un mode particulier de réalisation du procédé de concentration selon l'invention.FIG. 1 represents the different steps, for arriving at the concentration of a target DNA, according to a particular embodiment of the concentration method according to the invention.
La figure 2 représente une vue détaillée d'un film interstitiel créé lors de la formation d'une mousse.FIG. 2 represents a detailed view of an interstitial film created during the formation of a foam.
Les figures 3, 4 et 5 représentent des procédés de purification, de détection et de concentration mises en œuvre après le procédé de concentration explicité sur la figure 1.FIGS. 3, 4 and 5 represent purification, detection and concentration methods implemented after the concentration method explained in FIG. 1.
EXPOSE D'UN MODE PARTICULIER DE REALISATION DE L'INVENTION.PRESENTATION OF A PARTICULAR EMBODIMENT OF THE INVENTION
La figure 1 représente, à titre illustrâtif et non limitatif, la mise en œuvre, en quatre étapesFIG. 1 represents, by way of illustration and without limitation, the implementation, in four stages
(a), (b) , (c) et (d) , du procédé selon l'invention pour la concentration d'un ADN cible. L'étape (a) de la figure 1 représente un microbécher 10, dans lequel est déposé, à l'aide d'une micropipette ou d'une seringue, un échantillon liquide 12 contenant l'ADN cible 14, représenté sur la figure sous forme de brins. Ledit échantillon 12 est surmonté de l'atmosphère ambiante 11. Au cours de l'étape intitulée (b) sur la figure 1, une monocouche 16 comprenant un mélange de lipides ligands 18 et de lipides diluants 17 (rapport lipides ligants/lipides diluants 1 :4) est déposée à la surface de l'échantillon liquide 12. Cette monocouche 16 correspond, selon la terminologie de l'invention, à une couche interfaciale. Ces lipides ligands 18 sont tels que les ADN cibles vont pouvoir s'hybrider avec lesdits lipides. Pour cela, ces lipides doivent, au cours d'une étape préliminaire, être fonctionnalisés. Ainsi, les lipides ligands peuvent être des lipides initialement biotinylés, du type biotine- (LC) -DPPE, sur lesquels on adsorbé dans un premier temps de l'avidine puis dans un deuxième temps l'on greffe l'ADN complémentaire de l'ADN cible, sur l'avidine par le biais d'un groupement biotine fixé sur l'une des extrémités de l'ADN complémentaire. L'ensemble (lipide biotinylé-Avidine-ADN complémentaire biotinylé) constitue un lipide fonctionnalisé par une sonde permettant une hybridation spécifique des brins d'ADN cible.(a), (b), (c) and (d), of the method according to the invention for the concentration of a target DNA. Step (a) of FIG. 1 represents a microbeaker 10, in which is deposited, using a micropipette or syringe, a liquid sample 12 containing the target DNA 14, shown in the figure in the form of strands. Said sample 12 is surmounted by the ambient atmosphere 11. During the step entitled (b) in FIG. 1, a monolayer 16 comprising a mixture of lipid ligands 18 and lipid diluents 17 (ratio of lipid ligants / lipid diluents 1 : 4) is deposited on the surface of the liquid sample 12. This monolayer 16 corresponds, according to the terminology of the invention, to an interfacial layer. These lipid ligands 18 are such that the target DNAs will be able to hybridize with said lipids. For this, these lipids must, during a preliminary stage, be functionalized. Thus, the lipid ligands can be initially biotinylated lipids, of the biotin- (LC) -DPPE type, onto which avidin is first adsorbed and then, in a second step, the DNA complementary to the Target DNA, on avidin by means of a biotin group attached to one of the ends of the complementary DNA. The whole (biotinylated lipid-Avidin-complementary biotinylated DNA) constitutes a lipid functionalized by a probe allowing specific hybridization of the strands of target DNA.
Les lipides peuvent être également des lipides cationiques comprenant au moins une extrémité spermine, sur laquelle est adsorbëe un ADN complémentaire de l'ADN cible à concentrer. Il est bien entendu que les molécules aptes à fixer de l'ADN cible peuvent s'étendre à tout type de molécules aptes à fixer sélectivement l'ADN cible.The lipids can also be cationic lipids comprising at least one spermine end, on which is adsorbed a DNA complementary to the target DNA to be concentrated. It is understood that the molecules capable of fixing target DNA can extend to any type of molecules capable of selectively fixing the target DNA.
Conformément à l'invention et comme l'illustre l'étape (c) de la figure 1, une dispersion 20 du type mousse est créée par injection d'air dans l'échantillon liquide, ladite mousse étant constituée d'un ensemble de bulles, maintenu en cohésion par des films liquides interstitiels. La stabilité temporaire de la mousse est assurée par les lipides constitutifs de la phase sous forme de monocouche 16.In accordance with the invention and as illustrated in step (c) of FIG. 1, a foam-like dispersion 20 is created by injecting air into the liquid sample, said foam consisting of a set of bubbles , maintained in cohesion by interstitial liquid films. The temporary stability of the foam is ensured by the lipids constituting the phase in the form of a monolayer 16.
Une vue détaillée d'un film liquide interstitiel constitutif de la dispersion de type mousse est représentée sur la figure 2. Sur cette figure, ce film présente une forme octaédrique 22 de très faible volume, au sein duquel coexistent l'échantillon liquide 12 et la monocouche 16 ou couche interfaciale. De ce fait, l'ADN cible 14 se trouve quasiment en contact direct avec les lipides fonctionnalisés 18 de la monocouche ou couche interfaciale et se trouve ainsi très vite adsorbé par ces lipides 18. Le fait de passer par une mousse multiplie l'efficacité et la cinétique de capture de l'ADN par les lipides, du fait du très faible volume des films insterstitiels constitutifs de la mousse.A detailed view of an interstitial liquid film constituting the foam-type dispersion is shown in FIG. 2. In this figure, this film has an octahedral shape 22 of very small volume, within which the liquid sample 12 and the monolayer 16 or interfacial layer. As a result, the target DNA 14 is almost in direct contact with the functionalized lipids 18 of the monolayer or interfacial layer and is thus very quickly adsorbed by these lipids 18. The fact of passing through a foam multiplies the efficiency and the kinetics of DNA capture by lipids, due to the very small volume of the interstitial films constituting the foam.
Enfin, au cours d'une ultime étape représentée en (d) de la figure 1, la mousse est résorbée, laissant place à nouveau à un milieu biphasique non dispersé comprenant la monocouche de lipidique 24, de très faible épaisseur, au niveau de laquelle s'est adsorbé les ADN cibles sur les lipides hybrides 19, cette monocouche correspondant à une couche interfaciale référencée 24 sur le dessin et une phase 26 correspondant à l'échantillon liquide 12 appauvri en ADN cible. Il est bien entendu, que, sur l'ensemble de la figure 1, les lipides 17, 18 et 19 ne devraient être localisés uniquement qu'au niveau de la monocouche 16 ou couche interfaciale 24 (pour la figure ld) , d'épaisseur infime. Toutefois, pour des raisons de visibilité, lesdits lipides ont été considérablement grossis.Finally, during a final step represented in (d) of FIG. 1, the foam is resorbed, leaving again a non-dispersed biphasic medium comprising the lipid monolayer 24, of very small thickness, at the level of which adsorbed the target DNA on the hybrid lipids 19, this monolayer corresponding to an interfacial layer referenced 24 in the drawing and a phase 26 corresponding to the liquid sample 12 depleted in target DNA. It is understood that, on the whole of FIG. 1, the lipids 17, 18 and 19 should only be located at the level of the monolayer 16 or interfacial layer 24 (for FIG. 1d), of thickness tiny. However, for reasons of visibility, said lipids have been considerably enlarged.
Les figures 3, 4 et 5 illustrent différentes applications envisageables du procédé de concentration, mises en œuvre selon un mode de réalisation particulier décrit ci-dessus.Figures 3, 4 and 5 illustrate various possible applications of the concentration process, implemented according to a particular embodiment described above.
Ainsi, la figure 3 illustre le cas où, après concentration desdits ADN cibles, par le procédé explicité sur la figure 1, lesdits ADN sont détectés par des techniques de fluorescence. Dans ce cas, les lipides 19 fonctionnalisés par un ADN complémentaire de l'ADN cible et hybrides, comportent, en plus, un marqueur fluorescent. Ainsi, on peut déterminer la présence d'ADN cible au niveau de la couche interfaciale 24 par une mesure de fluorescence.Thus, FIG. 3 illustrates the case where, after concentration of said target DNAs, by the method explained in FIG. 1, said DNAs are detected by fluorescence techniques. In this case, the lipids 19 functionalized by a DNA complementary to the target DNA and hybrid, additionally contain a fluorescent label. Thus, the presence of target DNA can be determined at the interfacial layer 24 by a measurement of fluorescence.
La figure 4 illustre un cas particulier de purification d'un ADN préalablement concentré, par le procédé de concentration explicité selon la figure 1. Une fois que la résorption de la mousse est achevée, la phase 26 appauvrie en ADN, ledit ADN étant adsorbé, en majeure partie sur la couche 24, est soutirée à l'aide d'une micropipette 23 et l'on obtient - une couche interfaciale 24 d'ADN purifié.FIG. 4 illustrates a particular case of purification of a DNA previously concentrated, by the concentration process explained according to FIG. 1. Once the resorption of the foam is completed, phase 26 depleted in DNA, said DNA being adsorbed, mostly on layer 24, is withdrawn using of a micropipette 23 and one obtains - an interfacial layer 24 of purified DNA.
La figure 5 illustre le cas où le procédé de concentration est utilisé dans le seul but d'obtenir une phase plus concentrée en un ADN donné que la phase d'origine dudit ADN. A ce moment-là, la couche 24 riche en ADN cible est soutirée à l'aide d'une micropipette 23 pour être utilisée pour des applications diverses.FIG. 5 illustrates the case where the concentration method is used for the sole purpose of obtaining a phase more concentrated in a given DNA than the original phase of said DNA. At this time, the layer 24 rich in target DNA is withdrawn using a micropipette 23 to be used for various applications.
Il est bien entendu, que d'autres applications,, non représentées sur ces figures, peuvent être envisagées. It is understood that other applications, not shown in these figures, can be envisaged.

Claims

REVENDICATIONS
1. Procédé de concentration sélective d'une macromolécule ou d'un agglomérat de molécules ou de particules contenu dans un échantillon liquide, comprenant successivement les étapes suivantes :1. Process for the selective concentration of a macromolecule or an agglomerate of molecules or particles contained in a liquid sample, successively comprising the following steps:
- formation d'une dispersion stabilisée de type mousse ou emulsion, à partir d'un milieu comprenant ledit échantillon liquide et une couche interfaciale, ladite couche interfaciale étant apte à fixer sélectivement ladite macromolècule ou ledit agglomérat à concentrer ; et- Formation of a stabilized dispersion of foam or emulsion type, from a medium comprising said liquid sample and an interfacial layer, said interfacial layer being capable of selectively fixing said macromolecule or said agglomerate to be concentrated; and
- résorption de la dispersion formée lors de l'étape précédente de façon à reformer ladite couche interfaciale.- resorption of the dispersion formed during the previous step so as to reform said interfacial layer.
2. Procédé de concentration selon la revendication 1, dans lequel l'étape de formation de la dispersion est effectuée par agitation mécanique du milieu comprenant ledit échantillon liquide et ladite couche interfaciale.2. A concentration method according to claim 1, in which the step of forming the dispersion is carried out by mechanical agitation of the medium comprising said liquid sample and said interfacial layer.
3. Procédé de concentration selon la revendication 1, dans lequel l'étape de formation de la dispersion est effectuée par injection directement dans l'échantillon liquide de jets capillaires gazeux ou liquides .3. The concentration method according to claim 1, in which the step of forming the dispersion is carried out by injecting gaseous or liquid capillary jets directly into the liquid sample.
4. Procédé de concentration selon l'une quelconque des revendications 1 à 3, dans lequel la couche interfaciale comprend au moins une molécule apte à fixer sélectivement ladite macromolècule ou ledit agglomérat .4. Concentration method according to any one of claims 1 to 3, in which the interfacial layer comprises at least one suitable molecule to selectively fix said macromolecule or said agglomerate.
5. Procédé de concentration selon la revendication 4, dans lequel la molécule apte à fixer la macromolécule ou agglomérat de molécules ou de particules à concentrer est une molécule comportant des groupements aptes à fixer la macromolécule ou agglomérat par affinité chimique, polarisation électrique ou magnétique, et/ou ionisation, ladite molécule étant, de préférence, une molécule surfactante .5. A method of concentration according to claim 4, in which the molecule capable of fixing the macromolecule or agglomerate of molecules or particles to be concentrated is a molecule comprising groups capable of fixing the macromolecule or agglomerate by chemical affinity, electrical or magnetic polarization, and / or ionization, said molecule preferably being a surfactant molecule.
6. Procédé de concentration selon l'une quelconque des revendications l à 5, dans lequel la macromolécule est choisie parmi le groupe constitué par les acides nucléiques et les protéines, telles que les antigènes et les anticorps.6. Concentration method according to any one of claims 1 to 5, in which the macromolecule is chosen from the group consisting of nucleic acids and proteins, such as antigens and antibodies.
7. Procédé de concentration selon l'une quelconque des revendications 1 à 3, dans lequel l'agglomérat de molécules est un prion.7. A method of concentration according to any one of claims 1 to 3, wherein the agglomerate of molecules is a prion.
8. Procédé de concentration selon l'une quelconque des revendications 1 à 5, dans lequel l'agglomérat de particules est choisi parmi le groupe constitué de particules colloïdales.8. A concentration method according to any one of claims 1 to 5, in which the agglomerate of particles is chosen from the group consisting of colloidal particles.
9. Procédé selon l'une quelconque des revendications 1 à- 6, dans lequel la macromolécule à concentrer est l'ADN. 9. Method according to any one of claims 1 to 6, in which the macromolecule to be concentrated is DNA.
10. Procédé selon la revendication 4, dans lequel, lorsque la macromolécule à concentrer est l'ADN, la molécule apte à fixer l'ADN est fonctionnalisée par une sonde de façon à permettre l'hybridation spécifique de l'ADN à concentrer.10. The method of claim 4, wherein, when the macromolecule to be concentrated is DNA, the molecule capable of fixing the DNA is functionalized by a probe so as to allow specific hybridization of the DNA to be concentrated.
11. Procédé selon la revendication 10, dans lequel la molécule apte à fixer l'ADN est un lipide fonctionnalisé par une sonde ADN complémentaire de l'ADN à concentrer.11. The method of claim 10, wherein the molecule capable of fixing the DNA is a lipid functionalized by a DNA probe complementary to the DNA to be concentrated.
12. Procédé selon la revendication 11, dans lequel le lipide est un lipide biotinylé comportant un groupement avidine ou dérivé de l'avidine, sur lequel est greffé l'ADN complémentaire par une extrémité biotinylëe précédemment incorporée audit ADN.12. The method of claim 11, wherein the lipid is a biotinylated lipid comprising an avidin group or derivative of avidin, onto which the complementary DNA is grafted by a biotinylated end previously incorporated into said DNA.
13. Procédé selon la revendication 11, dans lequel le lipide est un lipide cationique comprenant au moins un groupement spermine sur lequel est adsorbé l'ADN complémentaire.13. The method of claim 11, wherein the lipid is a cationic lipid comprising at least one spermine group on which the complementary DNA is adsorbed.
14. Procédé de purification d'une macromolécule ou d'un agglomérat de molécules ou particules contenu initialement dans un échantillon liquide, comprenant la concentration de ladite macromolécule ou dudit agglomérat au sein d'une couche par la mise en oeuvre du procédé selon l'une quelconque des revendications 1 à 13, l'élimination de l'échantillon liquide appauvri en ladite macromolécule ou ledit agglomérat, après l'étape de concentration. 14. Method for purifying a macromolecule or an agglomerate of molecules or particles initially contained in a liquid sample, comprising the concentration of said macromolecule or said agglomerate within a layer by implementing the method according to any one of claims 1 to 13, the elimination of the liquid sample depleted in said macromolecule or said agglomerate, after the concentration step.
15. Procédé de détection d'une macromolécule ou d'un agglomérat de molécules ou particules contenu initialement dans un échantillon liquide comprenant la concentration au sein d'une couche de ladite macromolécule ou dudit agglomérat par la mise en oeuvre du procédé selon l'une quelconque des revendications 1 à 13 et la détection de ladite macromolécule ou dudit agglomérat au sein de ladite couche par des techniques appropriées de détection.15. Method for detecting a macromolecule or an agglomerate of molecules or particles initially contained in a liquid sample comprising the concentration within a layer of said macromolecule or said agglomerate by implementing the method according to one any of claims 1 to 13 and detecting said macromolecule or said agglomerate within said layer by appropriate detection techniques.
16. Procédé d'amplification d'une macromolécule ou d'un agglomérat de molécules ou particules contenu initialement dans un échantillon liquide comprenant la concentration de ladite macromolécule ou dudit agglomérat au sein d'une couche par la mise en oeuvre du procédé selon l'une quelconque des revendications 1 à 13 et le remplacement dudit échantillon liquide, après l'étape de concentration de ladite macromolécule ou dudit agglomérat au sein de ladite couche, par un liquide comprenant des agents d'amplification, suivie de l'étape d'amplification au moyen desdits agents.16. Method for amplifying a macromolecule or an agglomerate of molecules or particles initially contained in a liquid sample comprising the concentration of said macromolecule or said agglomerate within a layer by implementing the method according to any one of claims 1 to 13 and the replacement of said liquid sample, after the step of concentrating said macromolecule or of said agglomerate within said layer, with a liquid comprising amplifying agents, followed by the amplification step by means of said agents.
17. Procédé d'amplification selon la revendication 16, dans lequel la macromolécule est un17. The amplification method according to claim 16, in which the macromolecule is a
ADN.DNA.
18. Procédé d'amplification selon la revendication 16, dans lequel l'agglomérat de molécules est un prion. 18. The amplification method according to claim 16, in which the agglomerate of molecules is a prion.
PCT/FR2003/000920 2002-03-25 2003-03-24 Method of concentrating macromolecules or agglomerates of molecules or particles WO2003080209A2 (en)

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DE60308338T DE60308338T2 (en) 2002-03-25 2003-03-24 PROCESS FOR CONCENTRATING MACROMOLECULES OR AGGLOMERATES FROM MOLECULES OR PARTICLES
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